alpha s-Plasmin inhibitor (alpha 2PI), one of the serine protease inhibitors in plasma, was expressed in baby hamster kidney (BHK) cell line. The expression vector was constructed with its genomic DNA and cDNA, and was transfected into BHK cells by the calcium phosphate method. The recombinant alpha 2PI which was secreted from the cells was estimated by SDS-PAGE to have a molecular mass of 67 kDa, which is indistinguishable from that of normal plasma alpha 2PI. The leader peptide of 12 amino acids was retained at the amino terminus of the recombinant alpha 2PI. This finding suggests that alpha 2PI has pre-pro type processing and the propeptide of 12 amino acids is not removed in BHK cells. This pro-alpha 2PI shows essentially the same inhibitory activity on plasmin and the same affinity for plasmin(ogen) as those of normal alpha 2PI. However, the cross-linking ability to fibrin is reduced to less than one-third of that of normal alpha 2PI. The cross-linking site is the glutamine residue located at the second position from the amino terminus of normal alpha 2PI. The conformational change of this region caused by the addition of the propeptide may have affected the cross-linking capacity of the inhibitor.

We prepared a novel recombinant tumor necrosis factor-alpha (TNF) mutant (mutant 471), in which 7 N-terminal amino-acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, and compared its biological activity with that of wild-type recombinant TNF. Mutant 471 had a 7-fold higher anti-tumor activity against murine L-M cells in vitro, and a higher binding activity to TNF receptors on L-M cells, than wild-type TNF. Furthermore, mutant 471 showed a higher anti-tumor effect on murine Meth A-HM tumors transplanted into BALB/c mice, with complete regression of the tumors being observed in the animals. The possible cachectin activity of mutant 471 was almost the same as that of wild-type TNF. The acute lethal toxicity of mutant 471 in beta-D-galactosamine-sensitized C3H/HeJ mice was 18 times lower than that of wild-type TNF. These results suggest that mutant 471 might be a more promising anti-cancer agent than wild-type TNF.

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